1. Field of the Invention
The present invention relates to vaccines against Campylobacter colonisation, to the use of Campylobacter proteins and anti-Campylobacter antibodies for the preparation of such vaccines and to methods for the preparation of such vaccines.
2. Description of Related Art
Bacteria of the genus Campylobacter are Gram-negative spiral shaped pathogenic bacteria, with a high motility and carrying a flagellum at one or both poles of the cell. Several Campylobacter species have been found. Campylobacter jejuni is very often found in poultry. Frequently Campylobacter coli and (to a lesser extent) the recently found Campylobacter hyoilei are found in pigs.
Of these, Campylobacter jejuni is the most frequently isolated Campylobacter species in association with human diarrhoea. It is becoming more and more evident that the number of Campylobacter infections in humans exceeds the number of Salmonella infections. (Griffiths et al., Journ. of Applied Bacteriology 1990, 69: 281-301, Walker et al., Microbiological reviews 1986, 50: 81-94, Butzler, J-P., ISBN 0-8493-5446-3, RIVM Report No. 216852002, Bilthoven, the Netherlands). It is difficult to avoid infection in humans with Campylobacter since, first of all, Campylobacter is a food borne zoonotic bacterium for which many animals, both wild and domestic, healthy or sick serve as a reservoir. In addition the bacterium has many different routes of transmission. Bacteria can survive in a dormant coccal form for several weeks on e.g. the surface of carcasses and in water. The bacterium can therefore easily be transmitted to man through direct contact with animals or by means of contaminated water or food, e.g. milk or meat. C. jejuni is present in many healthy animals, e.g. avian species such as turkey and chicken, cattle, sheep, horses and rodents. Chicken meat, an important nutrient source in many countries world-wide is known to be very frequently contaminated with Campylobacter (Shane (1992), S. M., Avian Pathology 21: 189-213). This is not only the case in developing countries but also in e.g. Europe. Campylobacter resides in the gut of poultry. Contamination of the meat frequently happens in the slaughterhouse when the intestinal tract, which is often heavily Campylobacter-contaminated, is removed from the animal. Contamination during slaughter is very difficult to avoid. In the Netherlands, about 50% of the chicken meat is contaminated, in spite of the high hygienic standards applied in meat industry. A recent overview of the Epidemiology of Campylobacter in poultry is given in the Thesis of C. M. Karssen, (ISBN 90-71463-72-9). As a result of this high contamination pressure, about 300,000 persons annually in the Netherlands only (total population 15.000.000) suffer from Campylobacter infection, caused by handling or eating undercooked poultry meat. These figures are not significantly different in other European countries. World-wide, annually more than 400.000.000 cases are estimated to occur (Pace et al., Vaccine 1998, 16: 1563-1574). Campylobacter causes enteric infections in humans, and occasionally more severe diseases like abortion, meningitis, apendicitis, and urinary tract infection. (Blaser et al., New Engl. J. Med. 1981, 305: 1444-1452, Butzler et al., Clinics in Gastroenterol. 1979, 8: 737-765). Also, severe neurologic complications such as Guillain-Barrxc3xa9 syndrome and Miller-Fisher syndrome are sometimes seen (Schwerer et al., 1995. , J. Endotox. Res. 2: 395-403 and Salloway et al., 1996, Infect. Immun. 64: 2945-2949). Diarrhoea due to Campylobacter jejuni is usually a self-limiting infection, lasting about 2-7 days. In young children, old people and immuno-compromised patients, the disease is not self-limiting and requires antibiotic treatment.
It is clear that, if a potential vaccine against Campylobacter for human use would be available, it could prevent humans from becoming infected. This would however require a standard vaccination comparable to vaccination against e.g. mumps and measles. This is evidently not practical. A more logical approach lies in avoiding the transmission from animal to man, specifically from poultry to man. The easiest way of doing this is by vaccinating poultry against Campylobacter infection. Vaccination of poultry (as well as human vaccination) has however turned out to be much more complicated than was initially expected. This is due to the fact that Campylobacter is, in spite of the fact that it colonises the gut, not pathogenic to poultry. Most vaccines tested are inactivated whole cell preparations, administered systemically or orally, sometimes in combination with adjuvants. In some cases colonisation of the gut could, to a certain extent, be decreased, but there are no examples of vaccines avoiding colonisation. Shedding of Campylobacter could not be stopped by any of these vaccines. Killed whole cell vaccines, if compared with subunit vaccines, have been considered the best candidates for a vaccine, because in principle they still possess all potential immunogenic determinants. Next to the development of whole cell vaccines, much effort has been put in the development of flagella-based subunit vaccines. Flagella have been recognised as the immunodominant antigen recognised during infection and numerous studies have suggested a role for this protein in protection (Martin et al., Inf. And Immun. 1989, 57: 2542-2546, Wenman et al., J. Clin. Microbiol. 1985, 21: 108-112). Flagella-less mutants are known not to colonise the gut, and they disappear from the infected animal within one or two weeks whereas the wild type bacterium remains present in the gut. Flagella are thus by far the most likely candidates for the preparation of a vaccine, especially since they seem to play a key role, if not the only role, in the colonisation of the gut. If colonisation could be prevented, that would be a first step in the elimination of contamination in poultry. Nevertheless, potential vaccines based upon the flagella of Campylobacter have not given an acceptable level of protection.
Next to active vaccination as described above, passive vaccination has been tested as a means of decreasing Campylobacter infection. Tsubokura et al (1997, Clin. Exp. Immunol. 108: 451-455) have orally administered antibodies against whole Campylobacter jejuni cells followed by challenge with Campylobacter jejuni. They claim a 1-2 log reduction in the number of bacteria found in the faeces of thus vaccinated chickens. All efforts made so far have not yet led to any vaccine, be it live, inactivated or on the basis of subunits, that is capable of significantly diminishing the level of colonisation and the amount of bacteria shedded in the faeces. It is clear, that there still is a need for a reliable and safe vaccine or alternative treatment.
In principle, there is no need to protect poultry against Campylobacter infection during their whole life span. They do not suffer from infection as explained above. Therefore, a treatment capable of diminishing the amount of bacteria and thus the infective pressure shortly before slaughter would be an efficient treatment for suppressing subsequent contamination of the meat during slaughter. And this in turn would prevent meat-transmitted human Campylobacter infection.
It is an objective of the present invention to provide a vaccine that is capable of both diminishing the level of colonisation and shedding, or even of eliminating Campylobacter from the caecum of poultry. This avoids Campylobacter-contamination of the meat during slaughtering and therefore avoids subsequent infection of humans.
It was surprisingly found now that a vaccine having these characteristics can be based upon antibodies against flagella-less mutants of Campylobacter. This is highly unexpected since, as mentioned above, flagella are considered to be the key protein involved in adherence and colonisation. Even more surprising, such a vaccine does diminish colonisation and shedding of wild type flagellated Campylobacter. This is the first time that a vaccine is reported that is even capable to eliminate Campylobacter from the ceca below the level of detection.